Microcapsules have been studied for their applications to various kinds of uses in many fields such as pharmaceuticals, pesticides, foods, adhesives, and liquid crystals. For instance, in the field of pharmaceuticals, the microcapsules have been studied for their applications as sustained release pharmaceutical preparations by improving drugs used to be short in drug effect duration so as to exert their effects for long time. In addition to the persistence of pharmacological effects, expectations have been placed on a reduction in amount of a drug used, a reduction in side effect, an improvement in noncompliance, and so on. In recent years, furthermore, various release-controlled pharmaceutical compositions, which can release a drug at constant rates and have substantially zero-order drug-releasing rates, have been particularly proposed as sustained release pharmaceutical compositions. Those release-controlled agents, such as oral formulations, injectable formulations, and skin patch formulations, are in the process of developing.
In addition, for example, in the field of cosmetics as well as medical and pharmaceutical fields, the microcapsules have been expected to be materials that selectively transfer active ingredients having troubles in stability to the affected areas and permit their sustained release. Furthermore, for example, pesticides and fertilizers having sustained-release functions have been studied in the field of agriculture and also the application of various kinds of capsule ink has been studied in the field of recording materials.
In the field of pharmaceuticals, the specification of U.S. Pat. No. 614,665 discloses a method of manufacturing a pharmaceutical composition as a composition formed as a drug-encapsulating capsule using polyhydroxyalkanoate, in the form of fine particles in which a hydrophilic drug is entrapped in porous granules made of polyhydroxyalkanoate, or oil drops dissolving a lipophilic drug as a core material and encapsulated in a shell.
Among them, oral formulations have been extensively studied and developed and many pharmaceutical preparations have been placed on the market. On the other hand, regarding injectable formulations, insulin depot preparations have been partly used in the medical field. The reasons thereof include no development of a high molecular compound for imparting the ability of sustained release. The high molecular compounds used for oral formulations do not necessarily have to be decomposed in the living body. On the other hand, the decomposition, metabolism, and excretion of those for injectable formulations without the expression of toxicity in the living body are substantially indispensable prerequisites. Besides, there is a need of severe conditions of which, for example, no local disorder should be caused at the administration site.
Under such circumferences, many high molecular compounds have been studied in recent years. Among them, a polylactic acid, a lactate/glycolate copolymer, a hydroxybutyrate/glycolate copolymer, and so on, which are used for suture in an operation have been expected to be safe and useful high molecular compounds (JP 01-057087 B, WO94/10982, JP 08-151322 A, and JP 08-217691 A). Actually, for the purpose of preparing sustained release pharmaceutical preparations, many micro-encapsulation technologies using those high molecular compounds have been reported. In addition, with respect to poly-3-hydroxybutyrate (hereinafter, occasionally abbreviated as PHB), a microcapsule for a regulatory peptide from which the discharge of an active ingredient is controlled and a microcapsule containing Lastet have been reported (JP 61-431119 A, Drug Delivery System, 7(5), 367-371, 1992, and the same 8(2), 131-136, 1993). Furthermore, with respect to a 3-hydroxybutyrate/4-hydroxybutyrate copolymer, a sustained release pharmaceutical preparation where the rate of releasing a physiologically active substance is controlled by a monomer unit ratio has been disclosed (JP 11-199514 A).
Most of those technologies include water-soluble drugs. For instance, JP 60-100516 A and JP 62-201816 A each disclose a method of manufacturing a sustained-release microcapsule of a water-soluble drug having good dispersing qualities at a high trap rate by a underwater dry process. In addition, JP 01-158529 A and JP 02-124814 A each disclose a method of including a water-soluble drug in a polylactate/glycolate copolymer. Furthermore, a physiologically active polypeptide-containing sustained release pharmaceutical preparation is disclosed in JP 03-032302 A, an EGF-containing sustained release pharmaceutical preparation is disclosed in JP 02-330741 A, and disclosed in JP 04-321622 A is a long term sustained-release microcapsule that contains a copolymer or homopolymer of 7,000 to 30,000 in weight average molecular weight at a lactate/glycolate composition rate of 80/20 to 100/0 and performs zero-order release of a polypeptide for two or more months.
In this way, the conventional methods for manufacturing microcapsules can be grouped into three methods: a chemical method such as an interfacial polymerization method or an in-situ polymerization method; a physicochemical method such as a phase separation method (a coacervation method), an interfacial precipitation method, a submerged dry method, or an orifice method; and a mechanical method such as a spray drying method or a dry mixing method. Among them, interface polymerization method, in-situ polymerization method, submerged drying method, orifice method, phase-separation method (coacervation method), and so on have been proposed to be adopted as a method of micro-encapsulating the water-soluble drug.
There are many reports about sustained-release microcapsules of various physiologically active polypeptides and low-molecular water-soluble drugs (Critical Reviews in Therapeutic Drug Carrier Systems), vol. 12, pages 1-9, 1995; JP 02-503315 A; EPA 0586238; J. Pharm. Sci., vol. 75, pages 750-755 (1986); and JP 57-118512 A). At present, most of them cannot attain satisfactory long-term sustained release depending on their uses because: (1) a drug is encapsulated at a low rate because the drug leaks into an external water phase at a high rate in the manufacturing step; (2) the resulting capsules are generally porous and release a large quantity thereof at an initial stage; (3) a sufficient biological utilization factor cannot be obtained because a physiologically active substance is modified in the manufacturing step; and so on.
Regarding an improvement in sustained release of microcapsule, for the purpose of preventing a decrease in rate of releasing active ingredients after passing a predetermined time from the administration of a microcapsule in which polylactate is provided as a base material, JP 61-063613 A describes that fat-soluble additives (such as medium-chain fatty acid triglyceride and lower fatty acid triglyceride), which can be dissolved in a polylactate organic solvent and digested in the living body, are uniformly dissolved in the solvent solution. However, there is no suggestion about the application to other base materials and the preparation of a microcapsule using an aqueous solution of active ingredients. JP 08-151321 A discloses a microcapsule that contains an amorphous water-soluble physiologically active substance and a high molecular polymer and manufactured from an S/O/W type emulsion. However, there is no description with respect to a method of manufacturing a microcapsule using an aqueous solution of a drug as an internal water phase and a method using a metal complex of a water-soluble physiologically active peptide. Furthermore, EP 0765660 describes a microcapsule that contains an amorphous 2-pyperazinone-1-acetate derivative, and an S/O/W type emulsion is used in its manufacture. However, there is no description about a method of manufacturing a microcapsule in which an aqueous solution of a drug is used as an internal water phase and a method of using a metal complex of a water-soluble physiologically active peptide. Generally, in the manufacture of a microcapsule of a water-soluble physiologically active substance, the W/O type is superior in terms of uniformity and operability of the drug content to the S/O type in which a drug is used in a solid state. In industrial scale mass production, it is desired to use the W/O type.
In this way, a problem which is often pointed out in drug-releasing control using a sustained release pharmaceutical preparation is the presence of a phenomenon (an initial burst phenomenon) in which a large amount of the drug compound is released at once at the initial stage of releasing the drug after the administration of the sustained release pharmaceutical preparation into the body. The occurrence of the initial burst of the sustained release pharmaceutical preparation may cause the drug compound concentration in blood to exceed its acceptable level in the living body to thereby jeopardize the patient. A method of avoiding the initial burst to some extent by, for example, selecting the type of the drug compound and the structure of a biodegradable polymer has been discovered. However, any basic solution to prevent the initial burst phenomenon has not been found yet. On the other hand, furthermore, it has been desired to include a drug compound in a microcapsule in as high a concentration as possible, for releasing the drug compound for long time, or for including an expensive drug in a small amount of the pharmaceutical preparation as cost-effectively as possible.
However, in the conventional method of preparing microcapsules, the proportion (uptake rate) of a drug compound taken within a microcapsule tended to be low. In particular, when a water-soluble drug was used as the drug, there was a large problem in that the encapsulation rate of the drug was low because the drug was easy to scatter out of a membrane. In addition, a microcapsule prepared by a method that would allow an increase in uptake rate had a disadvantage in that an initial burst phenomenon tended to take place at the time of releasing the drug.
In addition, in the field of ultrasonic diagnosis or examination, it has been proposed to administer a microballoon, a miniature ball of a polymer, as an ultrasonic reflector in the body. Conventionally, it has been known that minute air bubbles dispersed in a liquid, i.e., micro-bubbles, are ultrasonic reflectors extremely effective in ultrasonic diagnosis or examination. However, the micro-bubbles disappear in the shortest possible time, or within minutes even when they are added with a stabilizer. Therefore, there is a need of administrating micro-bubbles in the body immediately after the preparation of the bubbles, so that the use thereof in the actual medical field has been difficult. In addition, after the administration in the body, for making the transmission of a bubble through a blood vessel easy, the size of the bubble must be in the range of about 1 to 10 μm. In micro-bubbles, most of bubbles formed are approximately 40 to 50 μm in size. In this respect, it has not been suitable for administering micro-bubbles in the living body to be utilized in ultrasonic diagnosis.
For solving the problems that the micro-bubbles involve, administration of a microballoon which is a miniature ball of a polymer as described above in the living body has been proposed (e.g., JP 03-503684 A). However, the microballoon obtained by the conventional method should be administered in large quantities to obtain a higher cystographic effect (contrast effect). In particular, a problem was that there was no effective contrast agent that sufficiently satisfies a high cystographic effect (a contrast effect) being desired particularly in the case of contrasting the cardiac muscle. The factors thereof include difficulty in obtaining uniform fine particles that contain many air bubbles because they do not have hollow structures in their insides. Besides, the massive administration of microballoon may place excessive burdens on the living body. Thus, a problem which should be improved has remained from the point of view of safety.
Furthermore, because a capsule structure that contains a magnetic substance can be easily collected by magnetic force, mainly in the field of biochemistry, its excellent effects have been expected as a medical diagnostic drug carrier, a bacteria- or cell-separating carrier, a carrier for separating and purifying a nucleic acid or a protein, a drug delivery carrier, an enzyme reaction carrier, a cell culture carrier, and so on. Examples of a method of synthesizing a capsule structure that contains a magnetic substance include: a method in which a magnetic substance imparted with lipophilicity is dispersed in a polymerizable monomer and the dispersion is subjected to suspension polymerization (JP 59-221302 A); a method in which a magnetic substance imparted with lipophilicity is dispersed in a polymerizable monomer in the same way and the mixture is homogenized in water with a homogenizer and polymerized to obtain magnetic particles having comparatively small particle sizes (JP 04-03088 B); and a method in which a magnetic substance is introduced into the inside of porous polymer particles having a specific functional group by oxidizing an iron compound after the precipitation of the iron compound in the presence of the porous polymer particles to obtain magnetic particles having large particle sizes and uniformity in size (JP 05-10808 B).
However, when the capsule structures containing the magnetic substances obtained by those synthetic methods are used for medical diagnostic drug carriers or the like, even in the case where many magnetic substances are located inside the capsule structure, sensitivity may fall sharply, a nonspecific reaction may be shown, or the like. Thus, sufficient performance is hardly obtained in many cases. This is probably because the magnetic substance component may be eluted to impair practical performance as the magnetic substance is partially exposed on the surface of the capsule structure containing the magnetic substance or a micropass is formed between the surface of the structure and the magnetic substance in the inside thereof. In general, the hydrophilicity of a magnetic substance is higher than that of polymer particles. In the conventional synthetic process, the localization of a magnetic substance on the surface of a capsule structure or the periphery of the surface may be one of the great causes which spoil practical performance. Thus, the conventional magnetic substance-containing capsule structure is difficult to prevent the exposure of the contained magnetic substance component on the surface of the structure and the elusion of the magnetic substance component by the formation of a micropass or the like. Therefore, the actual condition was that the conventional capsule structure was only limited to be used in the field where the elusion was insignificant.
By the way, in recent years, the production of a high molecular compound by means of biotechnology has been actively studied and partially translated in practical applications. For instance, known high molecular compounds derived from microorganisms include: PHAs such as PHB and a copolymer of 3-hydroxy-n-butyrate and 3-hydroxy-n-valerate (hereinafter, occasionally abbreviated as PHB/V); polysaccharides such as bacterial cellulose and pullulan; and polyamino acids such as poly-γ-glutamate and polylysine. In particular, like the conventional plastics, PHA can be used in various products by melt processing and so on and is excellent in biocompatibility, so that application of PHA in a medical soft material or the like has been expected.
Up to now, it has been reported that many microorganisms produce PHAs and accumulate them into the microbial cells. The production of PHB/V by microorganisms, Alcaligenes eutrophus Strain H16 ATCC No. 17699, Methylobacterium sp., Paracoccus sp., Alcaligenes sp., and Pseudomonas sp., have been reported (JP 05-074492 A, JP 06-015604 B, JP 07-014352 B, and JP 08-019227 B).
In addition, there is disclosed that Comamonas acidovorans Strain IFO 13852 produces PHA having 3-hydroxy-n-butyrate and 4-hydroxy-n-butyrate as monomer units (JP 09-191893 A). Furthermore, there is disclosed that Aeromonas caviae produces a copolymer of 3-hydroxy-n-butyrate and 3-hydroxyhexanoate (JP 05-093049 A and JP 07-265065 A).
The biosynthesis of those PHB and PHB/V can be carried out by an enzymatic polymerization reaction using as a substrate (R)-3-hydroxybutyryl-CoA or (R)-3-hydroxyvaleryl CoA produced from various carbon sources through various metabolic pathways in the living body.
The enzyme that catalyzes the polymerization reaction is a PHB synthetic enzyme (also referred to as a PHB polymerase or a PHB synthase). Here, “CoA” is an abbreviation for “Coenzyme A” and the chemical structure thereof is as follows.

In addition, in recent years, researches have been extensively carried out with respect to polyhydroxyalkanoate composed of a 3-hydroxyalkanoate unit having a medium-chain-length of about 3 to 13 carbon atoms (occasionally abbreviated as mcl-PHA). JP 2642937 B discloses the production of PHA having a 3-hydroxyalkanoate monomer unit having 6 to 12 carbon atoms by the addition of a noncyclic aliphatic hydrocarbon to Pseudomonas oleovorans Strain ATCC 29347. Furthermore, Appl. Environ. Microbiol., 58, 746 (1992) reports the production of PHA by Pseudomonas resinovorans in which octanoic acid is used as a single carbon source and 3-hydroxy-n-butyrate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, or 3-hydroxydecanoate is used as a monomer unit, and also the production of PHA by Pseudomonas resinovorans in which hexanoic acid is used as a single carbon source and 3-hydroxy-n-butyrate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, or 3-hydroxydecanoate is used as a monomer unit. Here, a 3-hydroxyalkanoate monomer unit having a chain length longer than that of the fatty acid in a raw material may be introduced by way of a fatty acid synthesis pathway described later.
Int. J. Biol. Macromol., 16(3), 119 (1994) reports the production of PHA by Pseudomonas sp. strain 61-3 in which sodium gluconate is used as a single carbon source and a 3-hydroxyalkanoate such as 3-hydroxy-n-butyrate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, or 3-hydroxydodecanoate and a 3-hydroxyalkenoate such as 3-hydroxy-5-cis-decenoate or 3-hydroxy-5-cis-dodecenoate are used as units.
The above PHA is one that consists of a monomer unit having an alkyl group on its side chain (hereinafter, occasionally abbreviated as usual-PHA). However, in consideration of more wide-ranging applications, such as an application as a functional polymer, an extremely useful PHA is one in which a substituent, except an alkyl group (e.g., a phenyl group, unsaturated hydrocarbon, ester group, allyl group, cyano group, halogenated hydrocarbon, or epoxide), is introduced into the side chain (hereinafter, occasionally abbreviated as unusual-PHA).
As an example of the biosynthesis of unusual-PHA having a phenyl group, Macromolecules, 24, 5256-5260 (1991), Macromol. Chem., 191, 1957-1965 (1990), and Chirality, 3, 492-494 (1991) reports that Pseudomonas oleovorans produces PHA that contains a 3-hydroxy-5-phenyl valerate unit from 5-phenyl valerate. In addition, Macromolecules, 29, 1762-1766 (1996) reports that Pseudomonas oleovorans produces PHA that contains a 3-hydroxy-5-(4-tolyl) valerate unit from 5-(4-tolyl) valerate (5-(4-methylphenyl) valerate). Furthermore, Macromolecules, 32, 2889-2895 (1999) reports that Pseudomonas oleovorans produces PHA that contains a 3-hydroxy-5-(2,4-dinitrophenyl) valerate unit and a 3-hydroxy-5-(4-nitrophenyl) valerate unit from 5-(2,4-dinitrophenyl) valerate.
In addition, as an example of unusual-PHA having a phenoxy group, Macromol. Chem. Phys., 195, 1665-1672 (1994) reports that Pseudomonas oleovorans produces PHA that contains a 3-hydroxy-5-phenoxy valerate unit and a 3-hydroxy-9-phenoxy nonanoate unit from 11-phenoxy undecanoate. Furthermore, Macromolecules, 29, 3432-3435 (1996) reports that Pseudomonas oleovorans produces PHA that contains a 3-hydroxy-4-phenoxy butyrate unit and a 3-hydroxy-6-phenoxy hexanoate unit from 6-phenoxy hexanoate, PHA that contains a 3-hydroxy-4-phenoxy butyrate unit, a 3-hydroxy-6-phenoxy hexanoate unit, and a 3-hydroxy-8-phenoxy octanoate unit from 8-phenoxy octanoate, and PHA that contains a 3-hydroxy-5-phenoxy valerate unit and a 3-hydroxy-7-phenoxy heptanoate unit from 11-phenoxy undecanoate.
Furthermore, Can. J. Microbiol., 41, 32-43 (1995) reports that each of Pseudomonas oleovorans Strain ATCC 29347 and Pseudomonas putida Strain KT 2442 produces PHA that contains a 3-hydroxy-p-cyanophenoxy hexanoate unit or a 3-hydroxy-p-nitrophenoxy hexanoate unit from p-cyanophenoxy hexanoate or p-nitrophenoxy hexanoate. In addition, JP 2989175 B describes a homopolymer consisting of a 3-hydroxy-5-(monofluorophenoxy) valerate unit or of a 3-hydroxy-5-(difluorophenoxy) valerate unit, a copolymer containing at least a 3-hydroxy-5-(monofluorophenoxy)pentanoate unit or a 3-hydroxy-5-(difluorophenoxy)pentanoate unit, and their manufacturing methods.
Furthermore, as an example of unusual-PHA having a cyclohexyl group, Macromolecules, 30, 1611-1615 (1997) reports that Pseudomonas oleovorans produces such PHA from cyclohexyl butyrate or from cyclohexyl valerate.
Furthermore, among PHAs in which substituents are introduced into their side chains, as an example of the development of PHA having a sulfur atom in the form of sulfide (—S—) in the side chain, Macromolecules., 32, 8315-8318 (1999) reports the production of PHA that contains 3-hydroxy-5-(phenylsulfanyl) valerate and 3-hydroxy-7-(phenylsulfanyl) heptanoate as monomer units using Pseudomonas putida Strain 27N01 with octanoic acid and 11-(phenylsulfanyl)undecanoate as substrates. However, in that case, the method used involves: pre-incubating Pseudomonas putida Strain 27N01 in a culture that contains only octanoic acid as a grow substrate: and inoculating the medium of the above culture into a culture that contains only 11-(phenylsulfanyl)undecanoate as a substrate.
Furthermore, Polymer Preprints, Japan Vol. 49, No. 5, 1034 (2000) reports that the production of PHA containing 3-hydroxy-5-benzyl thiovalerate and 3-hydroxy-7-[(phenylmethyl)sulfanyl]heptanoate as monomer units using Pseudomonas putida Strain 27N01 with 11-[(phenylmethyl)sulfanyl]undecanoate as a substrate. However, in this case, the method used involves: pre-incubating Pseudomonas putida Strain 27N01 in a culture that contains only octanoic acid as a grow substrate; and inoculating the medium of the above culture into a culture that contains only 11-[(phenylmethyl)sulfanyl]undecanoate as a substrate.
The biosynthesis of those mcl-PHA and unusual-PHA is performed by enzymatic polymerization reactions, where the substrate used is (R)-3-hydroxyacyl CoA produced from various alkanoic acids used as raw materials through various metabolic pathways in the living body (e.g., a β-oxidation system and a fatty acid synthesis pathway). The enzyme that catalyzes such a polymerization reaction is a PHA-synthetic enzyme (also referred to as a PHA polymerase or PHA synthase). Here, with respect to the PHB synthetic enzyme described above, the monomer to serve as a substrate for the PHA synthetic enzyme is limited. The PHB synthetic enzyme belongs to the category of PHA synthetic enzyme.
Hereinafter, there will be described the reaction until PHA is produced from an alkanoic acid through a polymerization reaction with a β-oxidation system and a PHA synthetic enzyme.

On the other hand, in the case of passing through the fatty acid synthesis pathway, PHA may be similarly synthesized using the PHA synthetic enzyme using (R)-3-hydroxyacyl-CoA as a substrate, which is converted from (R)-3-hydroxyacyl-ACP (“ACP” denotes an acyl-carrier protein) generated in the pathway.
In recent years, it has been attempted to synthesize PHA in a cell-free system (in vitro) by taking the above PHB or PHA synthetic enzyme out of microbial cells. In Proc. Natl. Acad. Sci. USA, 92, 6279-6283 (1995), the synthesis of PHB consisting of a 3-hydroxy-n-butyrate unit is achieved by acting 3-hydroxybutyryl-CoA on a PHB synthetic enzyme derived from Alcaligenes eutrophus. In addition, in Int. J. Biol. Macromol., 25, 55-60 (1999), the synthesis of PHA consisting of a 3-hydroxy-n-butyrate unit or a 3-hydroxy-n-valerate unit is achieved by acting 3-hydroxybutyryl-CoA or 3-hydroxyvaleryl-CoA on a PHB synthetic enzyme derived from Alcaligenes eutrophus. Besides, in this report, PHA consisting only of the R-isomer of 3-hydroxy-n-butyrate unit can be synthesized by acting 3-hydroxybutyryl-CoA in the form of a racemic body by virtue of the stereoselectivity of the enzyme. In addition, Macromol. Rapid Commun., 21, 77-84 (2000) reports the extracellular synthesis of PHB using a PHB synthetic enzyme derived from Alcaligenes eutrophus. 
Furthermore, in FEMS Microbiol. Lett., 168, 319-324 (1998), the synthesis of PHB consisting of a 3-hydroxy-n-butyrate unit is achieved by acting 3-hydroxybutyryl-CoA on a PHB synthetic enzyme derived from Chromatium vinosum. 
In Appl. Microbiol. Biotechnol., 54, 37-43 (2000), PHA consisting of a 3-hydroxydecanoate unit is synthesized by acting 3-hydroxydecanoyl-CoA on a PHA synthetic enzyme from Pseudomonas aeruginosa. 